U.S. patent application number 09/908321 was filed with the patent office on 2002-08-29 for efficient method of synthesizing combretastatin a-4 prodrugs.
Invention is credited to Baldwin, Amy, Gale, Jonathan, Haider, Reem, Hoare, John, Seyedi, Faye.
Application Number | 20020119951 09/908321 |
Document ID | / |
Family ID | 22816425 |
Filed Date | 2002-08-29 |
United States Patent
Application |
20020119951 |
Kind Code |
A1 |
Seyedi, Faye ; et
al. |
August 29, 2002 |
Efficient method of synthesizing combretastatin A-4 prodrugs
Abstract
Methods of synthesizing a phosphate ester of combretastat-in A-4
and trans-isomers thereof in which combretastatin A-4 is reacted
with dibenzylphosphite in the presence of carbon tetrabromide, or
with 2,2,2-trichloroethyl phosphorodichloridate, to form a
phosphate ester of combretastatin A-4 with protecting groups
thereon.
Inventors: |
Seyedi, Faye; (Canton,
MA) ; Gale, Jonathan; (W. Townsend, MA) ;
Haider, Reem; (Lexington, MA) ; Hoare, John;
(Lunenburg, MA) ; Baldwin, Amy; (Belmont,
MA) |
Correspondence
Address: |
Christopher C. Dunham
c/o Cooper & Dunham LLP
1185 Avenue of the Americas
New York
NY
10036
US
|
Family ID: |
22816425 |
Appl. No.: |
09/908321 |
Filed: |
July 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60218766 |
Jul 17, 2000 |
|
|
|
Current U.S.
Class: |
514/62 ; 514/143;
514/79; 536/55.2; 558/89 |
Current CPC
Class: |
Y02P 20/55 20151101;
C07F 9/12 20130101 |
Class at
Publication: |
514/62 ; 514/79;
514/143; 558/89; 536/55.2 |
International
Class: |
C07F 009/02; A61K
031/675; A61K 031/66 |
Claims
What is claimed is:
1. A method of synthesizing a phosphate ester of combretastatin A-4
and trans-isomers thereof in which: combretastatin A-4 having the
following chemical structure 14is reacted with dibenzylphosphite in
the presence of carbon tetrabromide to form said phosphate ester of
combretastatin A-4 with protecting groups thereon.
2. A method of synthesizing a phosphate ester of combretastatin A-4
and trans-isomers thereof in which: combretastatin A-4 having the
following chemical structure 15is reacted with 2,2,2-trichloroethyl
phosphorodichloridate forming said phosphate ester of
combretastatin A-4 with protecting groups thereon.
3. The method according to claim 2 wherein said
2,2,2-trichloroethyl phosphorodichloridate is reacted in the
presence of triethylamine.
4. A method of synthesizing a phosphoric acid of combretastatin A-4
and trans-isomers thereof in which: a phosphate ester of
combretastatin A-4 with protecting groups thereon having the
following chemical structure 16is reacted with bromotrimethylsilane
to form said phosphoric acid of combretastatin A-4.
5. A method of synthesizing combretastatin A-4 prodrugs and
trans-isomers thereof as phosphate salts comprising: reacting
combretastatin A-4 having the following chemical structure 17with
an activated phosphorylating agent having hydroxyl-protecting
groups thereon wherein said phosphorylating agent is either
dibenzylphosphite/carbon tetrabromide or 2,2,2-trichloroethyl
phosphorodichloridate, to form a phosphate ester of combretastatin
with protecting groups thereon; deprotecting said
hydroxyl-protecting groups with a deprotecting agent to yield a
phosphoric acid of combretastatin A-4; and reacting said phosphoric
acid with reactive agent to form a phosphate salt of combretastatin
A-4.
6. The method according to claim 5 wherein the deprotecting agent
is bromotrimethylsilane when said phosphorylating agent is
dibenzylphosphite/carbon tetrabromide.
7. The method according to claim 5 wherein Zn/Cu amalgam is the
deprotecting agent when said phosphorylating agent is
2,2,2-trichloroethyl phosphorodichloridate.
8. The method according to claim 5 wherein said phosphoric acid is
reacted with sodium methoxide to form a disodium phosphate salt or
a monosodium phosphate salt of combretastatin A-4.
9. The method according to claim 5 wherein the said reactive agent
is either alkaline metal or inorganic salt.
10. The method according to claim 5 wherein said reactive agent
forms X-phosphate salt of combretastatin A-4, wherein X is selected
from the group consisting of sodium, cesium, calcium, lithium,
magnesium, manganese, potassium, zinc, imidazole, morpholine,
piperazine, piperidine, pyrazole, pyridine, adenosine, cinchonine,
glucosamine, quinine, quinidine, tetracycline, verapamil.
11. The method of synthesizing combretastatin A-4 prodrugs and
trans-isomers thereof comprising: dissolving combretastatin A-4 in
acetonitrile to form a first solution; admixing triethylamine and
carbon tetrabromide in said first solution to form a second
solution; dissolving dibenzylphosphite in acetonitrile to said
second solution to form a third solution; admixing said third
solution to said second solution to form a fourth solution; and
admixing bromotrimethylsilane to said fourth solution and treating
with sodium methoxide in methanol to form a fifth solution of
phosphate salt of combretastatin A-4.
12. The method according claim 11 wherein said phosphate salt is
either a monosodium phosphate salt or a disodium phosphate salt of
combretastatin A-4.
13. The method according to claim 11 further comprising isolating
the phosphate salt of combretastatin A-4 from said fifth solution
to form a crude product; suspending said crude product in H.sub.2O
to form a sixth solution; treating said sixth solution with sodium
methoxide in methanol to form a basic solution; heating said basic
solution to about 35-40.degree. C.; and admixing acetone to cause
said phosphate salt of combretastatin A-4 to recrystalize from said
basic solution.
14. In a method of preparing combretastatin A-4 prodrugs and
trans-isomers thereof by forming a reaction mixture of
combretastatin A-4 with a phosphorylating agent to form a phosphate
ester with protecting groups thereon, cleaving said protective
groups with a deprotecting agent to form a phosphoric acid
derivative of combretastatin A-4 and treating said phosphoric acid
with a reactive agent to form a phosphate salt of combretastatin
A-4, the improvement of said method wherein: said phosphorylating
agent is either dibenzylphosphite/carbon tetrabromide or
2,2,2-trichloroethyl phosphorodichloridate; the deprotecting agent
is bromotrimethylsilane when said phosphorylating agent is
dibenzylphosphite/carbon tetrabromide and Zn/Cu amalgam when said
phosphorylating agent is said 2,2,2-trichloroethyl
phosphorodichloridate; and said reactive agent forms X-phosphate
salt of combretastatin A-4, wherein X is selected from the group
consisting of sodium, cesium, calcium, lithium, magnesium,
manganese, potassium, zinc, imidazole, morpholine, piperazine,
piperidine, pyrazole, pyridine, adenosine, cinchonine, glucosamine,
quinine, quinidine, tetracycline and verapamil.
15. The method according to claim 14 further comprising reacting
said reaction mixture with triethylamine to form said phosphate
ester when the phosphorylating agent is 2,2,2-trichloroethyl
phosphorodichloridate.
16. The improvement method according to claim 14 wherein said
X-phosphate salt of combretastatin is disodium phosphate.
17. In a method of preparing combretastatin A-4 prodrugs and
trans-isomers thereof by treating combretastatin A-4 with a
phosphorylating agent to form a phosphate ester of combretastatin
A-4 with protecting groups thereon, cleaving said protective groups
with a deprotecting agent to form phosphoric acid derivative of
combretastatin A-4 and treating said phosphoric acid with an agent
to form a salt of combretastatin A-4 phosphate, the improvement of
said method wherein: said combretastatin A-4 is dissolved in
acetonitrile, triethylamine and carbon tetrabromide to form a first
solution; adding to said first solution the phosphorylating agent
dibenzylphosphite to form a second solution comprising said
phosphate ester; said second solution is treated with the
deprotecting agent bromotrimethylsilane to form a phosphoric acid
solution; and said phosphoric acid solution is treated with a
reactive agent to form X-phosphate salt of combretastatin A-4,
wherein X is selected from the group consisting of sodium, cesium,
calcium, lithium, magnesium, manganese, potassium, zinc, imidazole,
morpholine, piperazine, piperidine, pyrazole, pyridine, adenosine,
cinchonine, glucosamine, quinine, quinidine, tetracycline and
verapamil.
18. The method according to claim 17 wherein said X-phosphate salt
of combretastatin is disodium phosphate to form a disodium
phosphate salt of combretastatin A-4.
19. The method according to claim 17 wherein said reactive agent is
treated with sodium methoxide to form a disodium phosphate salt of
combretastatin A-4.
20. The method according to claim 17 wherein said reactive agent is
sodium methoxide to form a basic solution.
21. The method according to claim 17 wherein said basic solution
has a pH of about 10-12.
22. The method according to claim 17 wherein said basic solution is
further cooled to recrystallize the phosphate salt of
combretastatin A-4.
23. A method of synthesizing a combretastatin A-4 prodrug,
comprising: (a) obtaining a phosphonium salt of
3,4,5-trimethoxybenzyl bromide by mixing a brominating reagent and
3,4,5-trimethoxybenzyl alcohol in toluene to obtain said bromide,
and adding triphenylphosphine thereto; (b) obtaining tritylated
isovanillin by mixing an amine base, isovanillin, and trityl
chloride in an ether solvent, and after quenching, adding heptane
and ethyl acetate; (c) mixing a suspension of said phosphonium salt
in tetrahydrofuran, an alkyl lithium reagent, and a slurry of said
tritylated isovanillin, to obtain a cis/trans stilbene; (d)
reacting said cis/trans stilbene with an acid to obtain a product
consisting essentially of cis combretastatin A-4; and (e)
synthesizing a combretastatin A-4 prodrug by reacting said cis
combretastatin A-4 with an activated phosphorylating agent having
hydroxyl-protecting groups thereon wherein said phosphorylating
agent is either dibenzylphosphite/carbon tetrabromide or
2,2,2-trichloroethyl phosphorodichloridate, to form a phosphate
ester of combretastatin with protecting groups thereon;
deprotecting said hydroxyl-protecting groups with a deprotecting
agent to yield a phosphoric acid of combretastatin A-4; and
reacting said phosphoric acid with reactive agent to form a
phosphate salt of combretastatin A-4.
24. A method according to claim 23, wherein the brominating reagent
in step (a) is phosphorus tribromide.
25. A method according to claim 23, wherein the triphenylphosphine
in step (a) is unsubstituted triphenylphosphine.
26. A method according to claim 23, wherein the amine base in step
(b) is triethyl amine.
27. A method according to claim 23, wherein the solvent in step (b)
is tetrahydrofuran.
28. A method according to claim 23, wherein the trityl chloride in
step (b) is unsubstituted trityl chloride.
29. A method according to claim 23, wherein the alkyl lithium
reagent in step (c) is n-butyl lithium.
30. A method according to claim 23, wherein the acid in step (d) is
hydrochloric acid.
31. A method according to claim 23, wherein the phosphorylating
agent in step (e) is dibenzylphosphite/carbon tetrabromide.
32. A method according to claim 23, wherein the brominating reagent
in step (a) is phosphorus tribromide; wherein the
triphenylphosphine in step (a) is unsubstituted triphenylphosphine;
wherein the amine base in step (b) is triethyl amine; wherein the
solvent in step (b) is tetrahydrofuran; wherein the trityl chloride
in step (b) is unsubstituted trityl chloride; wherein the alkyl
lithium reagent in step (c) is n-butyl lithium; wherein the acid in
step (d) is hydrochloric acid; and wherein the phosphorylating
agent in step (e) is dibenzylphosphite/carbon tetrabromide.
33. A method of synthesizing combretastatin A-4 prodrugs as
phosphate salts comprising reacting combretastatin A-4 with a
phosphite having the formula HPOY.sub.2 where Y is benzyl, tert
butyl, butyl, ethyl, isopropyl, methyl, phenyl or propyl, in the
presence of carbon tetrabromide, to form a phosphate ester of
combretastatin with protecting groups thereon; deprotecting said
hydroxyl-protecting groups with a deprotecting agent to yield a
phosphoric acid of combretastatin A-4; and reacting said phosphoric
acid with reactive agent to form a phosphate salt of combretastatin
A-4.
34. Combretastatin A-4 disodium phosphite produced by the method of
claim 33 wherein said phosphite is dibenzyl phosphite, said
deprotecting agent is bromotrimethylsilane, and said reactive agent
reacts with said phosphoric acid to form a disodium phosphate salt
of combretastatin A-4.
35. Combretastatin A-4 disodium phosphite produced by the method of
claim 32 wherein said deprotecting agent is bromotrimethylsilane,
and said reactive agent reacts with said phosphoric acid to form a
disodium phosphate salt of combretastatin A-4.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the priority benefit, under 35
U.S.C. .sctn.119(e)(1), of applicants' copending U.S. provisional
application No. 60/218,766, filed Jul. 17, 2000, the entire
disclosure of which is incorporated herein by this reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to the field of
compounds with antiangiogenesis effects that may be useful in the
treatment of one or more neoplastic diseases.
[0003] In particular, the present invention relates to new and
efficient methods of synthesizing prodrugs of the known
anti-angiogenesis compound denominated combretastatin A-4 and its
analogs as described in U.S. Pat. Nos. 4,940,726; 5,409,953; and
5,569,786. More particularly, this invention relates to the
improved and efficient phosphorylation and deprotection of phenol
combretastatin A-4 in the synthesis of water soluble
antiangiogenesis prodrugs of combretastatin A-4.
[0004] Combretastatin A-4 (Formula 1 below) is reported to be an
antineoplastic compound inhibiting cancer cell growth and tubulin
assembly. 1
[0005] It is believed that combretastatin A-4 attacks the lining of
blood vessels that grow around tumors, thereby severing the blood
supply to the cancerous tumor. Although combretastatin A-4 has
exhibited strong anti-cancer activity, its development has been
inhibited by extremely poor solubility in water making development
and biological distribution impracticable.
[0006] Water-soluble prodrug derivatives of combretastatin A-4 have
been reported recently. In particular, synthesis of phosphate salts
of combretastatin A-4, designated "combretastatin A-4P" (Formula 2
below) have been found to impart the requisite water solubility to
the prodrug and are disclosed in U.S. Pat. No. 5,561,122 issued to
G. R. Pettit et al. on Oct. 1, 1996. The phosphate group of the
prodrug combretastatin A-4P reportedly is hydrolyzed in vivo to
liberate the active drug combretastatin A-4. However, the currently
disclosed methods for synthesizing combretastatin A-4P are
difficult, require the use of undesirable solvents or restricted
solvents, and are not easily scalable. 2
[0007] where X=H(Z) (monovalent) or X=Z (divalent),
[0008] Z=Na.sup.2+, Na.sup.+, Li.sup.+, Mg.sup.2+, Mn.sup.2+,
Zn.sup.2+, Ca.sup.2+, Cs.sup.2+, imidazole, morpholine, piperazine,
piperidine, pyrazole, pyridine, adenosine, cinchonine, glucosamine,
quinine, quinidine, tetracycline, verapamil.
[0009] An improved method of preparing prodrugs of combretastatin
is,necessary in order to meet the demand for an efficient and
scalable synthesis to produce combretastatin A-4P and isomers
thereof for effective use in treating cancer tumors and similar
diseases.
SUMMARY OF THE INVENTION
[0010] It is an object of the present invention to synthesize
prodrugs of combretastatin A-4 that are both water soluble and
stable. It is a further object of the invention to develop an
efficient and scalable method for synthesizing cis-and
trans-prodrugs of combretastatin A-4.
[0011] Although combretastatin A-4 is a potent anticancer agent,
its poor water solubility has hindered development of the drug as
an anticancer treatment. Current methods of synthesizing water
soluble derivatives of combretastatin A-4 require the use of
undesirable or restricted solvents, such as chloroform, pyridine,
dichloromethane or dimethylformamide ("DMF"), require extractions,
separations and dilution of the reaction solutions, and heating and
cooling of reaction mixtures at temperatures that are not suitable
for production of prodrugs of combretastatin A-4 in commercial
quantities.
[0012] As detailed herein, the subject invention provides a novel
and improved method of synthesizing combretastatin A-4P that
minimizes or eliminates the use of undesirable solvents, and
overcomes many other deficiencies of the prior art using a
continuous process. A novel process is herein disclosed in which
dibenzyl phosphite/carbon tetrabromide is used to phosphorylate
phenol combretastatin A-4 forming a phosphate ester of
combretastatin A-4 with benzyl protecting groups thereon. An
improved method of cleaving the benzyl protecting groups from the
phosphate ester of combretastatin A-4 is disclosed in which
bromotrimethylsilane is reacted with combretastatin A-4 to form
phosphoric acid of combretastatin. An alternate novel
phosphorylation process was concurrently developed and is herein
disclosed in which 2,2,2-trichloroethyl phosphorodichloridate
phosphorylates combretastatin A-4 to a phosphate ester with
trichloroethyl protecting groups thereon. The trichloroethyl groups
are then cleaved from combretastatin A-4 using Zn/Cu amalgam to
form a phosphoric acid of combretastatin A-4. Further improvements
to the current processes for synthesizing phosphate salts of
combretastatin A-4 are described herein disclosing a continuous
process that overcomes many obstacles and limitations to the use
and large scale production of combretastatin A-4 prodrugs.
[0013] In another aspect, the invention embraces the provision of
combretastatin A-4 prodrug products of the aforesaid novel and
improved method.
[0014] In a further aspect, the invention contemplates the
provision of methods of synthesizing combretastatin A-4 prodrugs
including a complete procedure for synthesizing cis combretastatin
A-4, to which the foregoing method steps and procedures may then be
applied to obtain the prodrug. The procedure for synthesizing cis
combretastatin A-4 in accordance with this aspect of the invention
includes the steps of obtaining a phosphonium salt of
3,4,5-trimethoxybenzyl bromide by mixing a brominating reagent and
3,4,5-trimethoxybenzyl alcohol in toluene to obtain the bromide,
and adding triphenylphosphine thereto; obtaining tritylated
isovanillin by mixing an amine base, isovanillin, and trityl
chloride in an ether solvent, and after quenching, adding heptane
and ethyl acetate; mixing a suspension of the phosphonium salt in
tetrahydrofuran, an alkyl lithium reagent, and a slurry of the
tritylated isovanillin, to obtain a cis/trans stilbene; and
reacting the cis/trans stilbene with an acid to obtain a product
consisting essentially of cis combretastatin A-4.
[0015] As in other embodiments of the invention, a combretastatin
A-4 prodrug may then be prepared from the latter product by
reacting the cis combretastatin A-4 with an activated
phosphorylating agent having hydroxyl-protecting groups thereon
wherein the phosphorylating agent is either
dibenzylphosphite/carbon tetrabromide or 2,2,2-trichloroethyl
phosphorodichloridate, to form a phosphate ester of combretastatin
with protecting groups thereon; deprotecting the
hydroxyl-protecting groups with a deprotecting agent to yield a
phosphoric acid of combretastatin A-4; and reacting the phosphoric
acid with reactive agent to form a phosphate salt of combretastatin
A-4.
[0016] In the combretastatin A-4 synthesis procedure described
above, it is currently preferred that the brominating reagent is
phosphorus tribromide, the triphenylphosphine is unsubstituted
triphenylphosphine, the amine base is triethyl amine, the ether
solvent is tetrahydrofuran, the trityl chloride is unsubstituted
trityl chloride, the alkyl lithium reagent is n-butyl lithium, and
the acid is hydrochloric acid.
[0017] Further features and advantages of the invention will be
apparent from the detailed description hereinafter set forth,
together with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a flow chart illustrating the first step in a
specific example of the complete synthesis of a combretastatin A-4
prodrug in an embodiment of the method of the invention;
[0019] FIG. 2 is a flow chart illustrating the second step in the
aforesaid specific example;
[0020] FIG. 3 is a flow chart illustrating the third step in the
same specific example;
[0021] FIG. 4 is a flow chart illustrating the fourth step in the
same specific example; and
[0022] FIG. 5 is a flow chart illustrating the fifth step in the
same specific example.
DETAILED DESCRIPTION
[0023] The elucidation and isolation of combretastatin A-4 are
described in U.S. Pat. No. 4,996,237 which issued to G. R. Pettit
et al., on Feb. 26, 1991, while early efforts to develop a
combretastatin A-4 prodrug are described in U.S. Pat. No.
5,561,122, which issued to G. R. Pettit on Oct. 1, 1996. The
general background information from each of those patents is
incorporated herein by reference. The subject invention presents a
novel method of synthesizing prodrugs of combretastatin A-4. More
specifically, the present invention provides novel methods of
phosphorylation and deprotection in the synthesis of prodrugs of
combretastatin A-4.
[0024] Troc Phosphorylation-Prior Synthetic Methods
[0025] Existing methods of preparing prodrugs of combretastatin A-4
using a 2,2,2-trichloroethyl phosphorodichloridate ("Troc")
protected phosphorylating group contain many deficiencies (the
"Troc Method"). While current Troc Methods vary slightly, a
detailed synthesis of combretastatin A-4 prodrug as illustrated in
Formula 3 is representative of a method of synthesizing
combretastatin A-4 using Troc Phosphorylation. The Troc Method
requires the use of neat pyridine, a toxic solvent with a high
boiling point making product isolation difficult, and requiring
stripping the solvent/reagent in the initial phosphorylation step.
Further, the Troc Method requires the use of carcinogenic
chloroform in the initial phosphorylation reaction. Moreover,
phosphorylation by this method requires the use of
dimethylformamide having a high boiling point of 153.degree. C. Use
of DMF necessitates the additional step of evaporating the solvent
during deprotection of the phosphoric ester. The Troc Method
requires the use of Zn/Cu amalgam to deprotect the intermediate of
the Troc group, leaving heavy metal contaminants that are difficult
to remove from the final product. Further steps that are involved
in the Troc Method include the use of an ion exchange column and
the subsequent evaporation of a large volume of solvent, extended
refrigeration for crystallization of intermediates, evaporation of
the solvents to dryness in both steps, chemical drying of the
chloroform solution of the protected phosphorylated product, and
isolation of the protected phosphorylated combretastatin A-4
prodrug product. Contributing to these time consuming and costly
steps, the Troc Method requires all reactions be performed at high
dilution and isolation by ion-exchange chromatography or similar
means, further increasing the time and cost of isolating the
combretastatin A-4 prodrug and its intermediates by this method and
limiting this method to small scale production. 3
[0026] The difficulties with existing phosphorylation methods in
the synthesis of combretastatin A-4P were investigated and a novel
was efficient synthesis of prodrugs of combretastatin A-4 was
developed that substantially reduced the cost and time required to
synthesize combretastatin A-4P. Table 1 summarizes the developments
that were made to improve upon the current phosphorylation methods
described above.
1TABLE 1 Summary of Improvements to Troc Phosphorylation Method
Entry Improvements Result 1 Replacement of pyridine with Reaction
proceeded faster triethylamine in phosphorylation and gave white
solid of combretastatin A-4P 2 Replace DMF with Acetonitrile 71%
crude yield Isolate intermediate Phosphate 46% recrystalization
Acid of combretastatin A-4 98.3 wt % Assay 3 No isolation of
intermediate 88% crude yield Phosphate Acid of combretastatin 41%
recrystalization A-4 95.5 wt % Assay 4 Recrystalization of
combretastatin Scaleable recrystalization A-4P from
acetonitrile/water developed 66% recovery from Entry 1 final
product
[0027] Novel Synthetic Method with Troc Phosphorylation
[0028] The first improvement to the existing Troc Method was the
replacement of neat pyridine with triethylamine ("TEA") and a
reactive amount of dimethylaminopyridine ("DMAP") (Table 1, Entry
1). The reaction proceeds much more rapidly with TEA than with
pyridine (1.5 hours vs. 16 hours). Replacing the DMF solvent
(boiling point of 152.8.degree. C.) with acetonitrile (boiling
point 82.degree. C.) (Table 1, Entry 2) was still a further
improvement making isolation of the product from solvent easier to
perform. The phosphate ester intermediate having Troc protecting
groups thereon may then be deprotected without the need for
isolation. Deprotection of the intermediate is performed using
acetonitrile in Zn/Cu amalgam to form the intermediate phosphate
acid of combretastatin A-4P.
[0029] The intermediate phosphate acid is isolated using a
Dowex.TM. ion-exchange resin, purchased from Aldrich Chemical
Company, Milwaukee, Wis., and thereby eliminating the need for
separation by chromatography. Alternatively, synthesis may be
performed without isolation of the intermediate phosphate acid
(Table 1, Entry 3) to produce product with low Zn/Cu levels (130
ppm) thereby limiting the cis/trans isomerization of intermediates
caused by such metals.
[0030] The improvements to the Troc phosphorylation method of the
subject invention overcome the problems attributable to the Troc
method disclosed in the prior art, resulting in a new and improved
phosphorylation method to synthesize the combretastatin A-4P using
Troc as a protecting group to form 3'-O-Bis-2,2, 2-(trichlorethyl)
phosphate combretastatin A-4 (5). See Formula 4. 4
[0031] Benzyl Phosphorylation-Prior Methods
[0032] An alternate phosphorylation method is described in
international patent application PCT/US99/00419, by Pettit, G. R.
et al. filed Jan. 8, 1999, describing the use of dibenzyl
phosphite/carbon tetrachloride to phosphorylate the phenol
combretastatin A-4 with benzyl protecting groups thereon and
deprotecting the intermediate using iodotrimethylsilane ("TMS-I")
(the "Benzyl-I Method") . See Formula 5. However, this method
requires the use of undesirable solvents and reagents such as
chloroform, chlorotrimethylsilane/sodium iodide, and
iodotrimethylsilane, which leave impurities that catalyze the
conversion of cis isomers of combretastatin A-4P to the trans
isomer resulting in product that is not optically pure. Further,
these undesirable solvents and reagents are highly toxic and use in
the synthesis necessitates lengthy heating and cooling reactions.
These as well as other problems with the Benzyl-I Method have been
overcome by the subject invention. 5
[0033] Novel Synthetic Method Using Dibenzyl Phosphite
Phosphorylation
[0034] Synthesis of combretastatin A-4P was further improved using
dibenzyl phosphite/carbon tetrabromide to phosphorylate the phenol
combretastatin A-4 (Formula 1) with benzyl protecting groups
thereon to form 3'-O-Bis(benzyl)phosphate combretastatin A-4. See
Formula 6.
[0035] Table 2 summarizes the improvements to the synthetic
processes of the prior art by use of the dibenzyl phosphite/carbon
tetrabromide phosphorylation method.
[0036] The combretastatin A-4 is phosphorylated using dibenzyl
phosphite in presence of triethylamine, carbon tetrabromide, and
DMAP, and dibenzylphosphite in acetonitrile to yield crude
3'-O-Bis(benzyl)phosphor- ylcombretastatin A-4. See (6) in Formula
6. These improvements to the benzyl phosphorylation reaction cause
the reaction to go to completion leaving only trace phenol
combretastatin (I). Further improvements to the process resulted in
the elimination of the use of DMAP in the reaction, which is a
difficult solvent to remove from the product due to its high
boiling point. The crude product is isolated and debenzylation of
3'-O-Bis(benzyl)phosphorylcombretastatin A-4 product is performed
using bromotrimethylsilane ("TMS-Br") in acetonitrile. The
capricious nature of the deprotection of the benzyl groups from the
phosphate ester was observed in Pettit Patent App. PCT/US99/00419.
Initial debenzylation experiments yielded only trans product as
determined by HPLC/UV analysis (Table 2, Entry 1). Addition of 0.1
eq of NaHCO.sub.3 gave a 50:50 mixture of the cis and trans product
(Entry 2). No improvement in the ratio of intermediate isomers was
noted when 1 equivalent of NaHCO.sub.3 was used (Entry 3). The
TMS-Br reaction with the addition of 1 equivalent of Hunnings base
gave only trans product (Entry 4). The TMS-I (stabilized with Cu
metal) reaction also gave only trans product (Entry 5). 6
2TABLE 2 Summary of Benzyl Phosphorylation Method Entry Method of
Improvements Result 1 TMS-Br (Aldrich)/Acetonitrile 98+% trans
isomer formed 2 TMS-Br/NaHCO.sub.3 0.1 eq 50/50 cis/trans 3
TMS-Br/NaHCO.sub.3 1 eq 50/50 cis/trans 4 TMS-Br/Hunnings Base
trans 5 TMS-I/Cu stabilized trans 6 Distilled TMS-Br cis 7
Distilled TMS-Br Shorter reaction time, no Continuous process
solvent evaporation 8 Dibenzyl route wt/wt 81.4%, Na 13%, KF
Continuous process 2.95%, recovery 76%
[0037] Hydrolysis of the combretastatin A-4 phosphate ester with
aqueous Na.sub.2CO.sub.3 gave no reaction. Since the HPLC of
3'-O-Bis(benzyl)phosphate combretastatin A-4 did not show the
presence of any trans isomer, the transformation from cis to trans
was apparently catalyzed by trace I.sub.2, Br.sub.2, or HBr
impurity. Using distillated TMS-Br (Aldrich, slightly orange) under
N.sub.2 atmosphere resulted in debenzylation of the phosphate ester
with no noted isomerization of the cis product (Entry 6). This
improvement to the debenzylation reaction overcame the need for
time-consuming and costly factions and recrystallization procedures
to isolate pure isomers of the phosphoric acid of combretastatin
A-4.
[0038] The phosphorylation and debenzylation steps were further
developed into a continuous process. Phenol combretastatin A-4 is
dissolved in acetonitrile and triethylamine ("TEA") and CBr.sub.4
is added. The reaction mixture is cooled to 0.degree. C. before
adding dibenzylphosphite in acetonitrile. The reaction proceeds for
approximately one hour. Completion of the reaction may be verified
by TLC and/or HPLC. Distilled bromotrimethylsilane is then added to
the same mixture. Colorless bromotrimethylsilane may be purchased
from Fluka for successful debenzylation of
3'-O-Bis(benzyl)phosphate combretastatin A-4. After debenzylation,
approximately 30-45 minutes to run reaction to completion, the
reaction is then quenched with a solution of 25w % sodium methoxide
in methanol and allowed to stir, preferably overnight. The crude
all cis product is filtered out (Entry 7) in approximately 75%
yield from cis-combretastatin A-4. In experimental results, the
reported w/w assay of the combretastatin A-4P product was 81.4%
desired (Entry 8). Since no impurities were observed in .sup.1H NMR
and HPLC it was concluded that the impurities were predominantly
inorganic salts.
[0039] In order to remove impurities, crude combretastatin A-4P may
be stirred into water/methanol mixture and the solution basified to
pH 10-12 resulting in the crude product to become completely
dissolved in solution. The mixture is then heated to " imately
35-40.degree. C. for about one hour.
[0040] Acetone is added to the solution and allowed to cool to room
temperature before a second volume of acetone was added. The
material is then stirred overnight and the product filtered out.
The experimental variations in solvent volume to gram of material
are described in Table 3. Optimal results were obtained by
recrystalizing the crude combretastatin A-4P material ("Product")
with a mixture of water/methanol/acetone (5/5/10 ml/g crude)
yielding in 40% recovery from starting retastatin A-4 (Entry
4).
3TABLE 3 Summary of Purification Methods Entry Improvements Result
1 Recrystalization of wt/wt 97.2%, pH 7.98, Na combretastatin A-4P
16%, KF 3.6%, Recovery water/methanol/ 48% acetone (mL/g solid)
4/4/8 2 Trituration of CA-4P wt/wt 98.1%, pH 7.59, Na 10%
H.sub.2O/Acetone 16%, KF 4.1%, Recovery 43% 3 Trituration of CA-4P
wt/wt 100.6%, pH 7.53, KF 20% H.sub.2O/Acetone 12%, Recovery 29% 4
Recrystalization of CA-4P wt/wt 98.8%, pH 8.65, Na water/methanol/
10.1%, KF 2.9%, Recovery acetone (mL/g solid) 5/5/10 40% 5
Recrystalization of CA-4P wt/wt 99.1%, pH 8.81, Na water/methanol/
10.1%, KF 5.28%, Recovery acetone (mL/g solid) 6/5/10 23% 6
Trituration of CA-4P wt/wt 98%, KF 2.87%, 20% H.sub.2O/Acetone
Recovery 43%
[0041] While the invention as described above embraces methods of
synthesizing prodrugs of combretastatin A-4 regardless of how the
combretastatin A-4 itself is obtained or prepared, in a further
sense the invention also contemplates complete methods of producing
combretastatin A-4 prodrugs including a preferred synthesis of
combretastatin A-4 followed by phosphorylation and deprotection to
provide the prodrug. An embodiment of such a complete method in
accordance with the invention will now be set forth.
[0042] Synthesis of Combretastatin A-4
[0043] Step 1 (Preparation of the Phosphonium Salt)
[0044] A cold solution of a brominating reagent in toluene is added
to a cold solution of 3,4,5-trimethoxybenzyl alcohol (TMBA) in
toluene and the mixture is stirred until the reaction is complete.
The brominating reagent currently preferred is phosphorus
tribromide (PBr.sub.3); examples of alternative brominating
reagents include gaseous HBr, triphenylphosphine dibromide and
SOBr.sub.2. The resulting bromide (TMBB) is quenched with water and
washed. The phases are separated and triphenylphosphine (Ph.sub.3P)
is added to the organic phase. As used herein, the term
"triphenylphosphine" includes unsubstituted triphenylphosphine,
which is currently preferred for this step, and singly or multiply
substituted triarylphosphines 7
[0045] in which the group(s) attached to the aryl ring(s) in the
phosphine may be lower alkyl, lower alkoxy, fluorine and nitro, the
substitution pattern on the ring(s) being any location other than
the carbon-phosphorus bond; the triphenylphosphine of Formula (7)
is unsubstituted when all R are H.
[0046] The mixture is stirred and the solid is collected, washed
and dried to provide the phosphonium salt, Compound I. Step 1 is
represented as follows: 8
[0047] and a flow chart of a specific example is shown in FIG.
1.
[0048] Step 2 (Trityl Protection of Isovanillin) An amine base is
combined with 3-hydroxy-4-methoxy-benzaldehyde (isovanillin),
triphenylmethyl chloride (trityl chloride or TrCl) and an ether
solvent, and the mixture is stirred with heating until the reaction
is complete. The amine base is preferably triethyl amine
(Et.sub.3N); more generally, the amine base may be a trialkyl amine
base (lower alkyl or cyclic, including aryl, up to six carbons per
alkyl group or ring, examples being Ph.sub.3N, R.sub.3N, and cyclic
amines such as pyridine, N-methyl morpholine, and DBU), or an amine
resin (such as polyvinyl pyridine or IRA-68 or equivalent). As used
herein, the term "trityl chloride" includes unsubstituted trityl
chloride, which is currently preferred for this step, and singly or
multiply (one to five groups) substituted aryl groups on the trityl
chloride; the group(s) attached to the aryl ring in the trityl
chloride may be lower alkyl, lower alkoxy, fluorine and nitro, the
substitution pattern on the ring being any location other than the
carbon-carbon bond forming the trityl chloride: 9
[0049] In formula (9), when R is H the trityl chloride is the
currently preferred unsubstituted trityl chloride. The ether
solvent may be lower alkyl or cyclic (including aryl) up to six
carbons per alkyl group or ring, the preferred solvent being
tetrahydrofuran (THF), other illustrative examples including
Et.sub.2O, dibutyl ether, methyl THF, MTBE, and dioxane. The
reaction is quenched with water, and a mixture of heptane and ethyl
acetate (EtOAc) is added. The mixture is stirred and the solid is
collected, washed and dried to provide the tritylated hydrovanillin
(Compound II). Step 2 is represented as follows: 10
[0050] and a flow chart of a specific example is shown in FIG.
2.
[0051] Step 3 (Preparation of Cis/Trans Product by Wittig
Reaction)
[0052] To a (preferably cold) suspension of the phosphonium salt
(Compound I) in THF is added n-butyl lithium (n-BuLi) followed by a
slurry of Compound II in THF. Alternatives to n-BuLi include other
alkyl amine bases such as methyl lithium, s-butyl lithium,
tert-butyl lithium, other commercially available alkyl lithium
reagents such as pentyl, hexyl and octyl lithium (available from
FMC LithCo Div), and hindered amine bases such as lithium
diisopropyl or dicyclohexyl amide and lithium hexamethyl
disilazane. The resulting mixture is stirred until the reaction is
complete. The reaction is quenched with brine at a cool temperature
and the phases are separated. The organic phase is partially
concentrated and diluted with ethanol. The resultant slurry is
stirred and cooled, and the product is collected, washed and dried
to provide a cis/trans-stilbene (Compound III), in which the ratio
of cis (Z) to trans (E) is 60-75% cis to 40-25% trans. Step 3 is
represented as follows: 11
[0053] and a flow chart of a specific example is shown in FIG.
3.
[0054] Step 4 (Detritylation Reaction)
[0055] A mixture of Compound III, acid (preferably hydrochloric
acid; alternatives include sulfuric acid, hydrobromic acid,
methanesulfonic acid, and acid resins such as amberlyst), and
toluene is stirred until the reaction is complete. The reaction is
quenched with water and the mixture is stirred with cooling. The
product is collected, washed and dried to provide exclusively the
cis-isomer of combretastatin A-4 (cis-CA4); Compound IV); i.e.,
only the cis-isomer crystallizes. Step 4 is represented as follows:
12
[0056] and a flow chart of a specific example is shown in FIG.
4.
[0057] Prodrug Preparation
[0058] Step 5 (Dibenzylphosphorylation Reaction, Deprotection and
Disodium Salt Formation)
[0059] A cold mixture of the cis-CA4 (Compound IV), a trialkyl
amine base (preferably Et.sub.3N), CBr.sub.4 and acetonitrile
(CH.sub.3CN) is combined with a mixture of dibenzyl phosphite
(HPO(OBn).sub.2) and CH.sub.3CN and the resulting mixture is
stirred at room temperature until phosphorylation is complete
(alternatives to dibenzyl phosphite include, e.g., di-tert butyl
phosphite, dibutyl phosphite, diethyl phosphite, diisopropyl
phosphite, dimethyl phosphite, diphenyl phosphite, and dipropyl
phosphite; together with dibenzyl phosphite, these may be
designated phosphites having the formula HPOY.sub.2 where Y is
benzyl, tert butyl, butyl, ethyl, isopropyl, methyl, phenyl or
propyl). Bromotrimethylsilane (TMSBr) is added and the mixture is
stirred until debenzylation is complete (alternatives to TMSBr
include, e.g., TMSCl/NaBr or NaI, and higher alkyl silyl bromides
up to four carbons per alkyl group) or the equivalent higher alkyl
silyl chlorides in conjunction with NaBr or NaI; the higher alkyl
silyl reagents will react much more slowly in this type of
reaction). The reaction is quenched with a solution of sodium
methoxide (NaOMe) in methanol (MeOH) and the mixture is stirred
(alternatives to NaOMe include, e.g., other sodium alkoxides such
as sodium ethoxide, isopropoxide, tert-butoxide and tert amyloxide;
sodium 2-ethyl hexanoate, sodium acetate or an ion exchange resin
that would act as a sodium carrier). The solid is collected, and
washed with acetone to provide a crude product. This crude product
is dissolved in a mixture of methanol and water with heat. The
solution is basified to pH 10-12 with methanolic sodium methoxide,
warmed and diluted with methanol and acetone. The solution is
cooled to room temperature; additional acetone is added; and the
product is collected and dried to provide the disodium salt of
combretastatin A-4 phosphate, CA4P (Compound V). Step 5 is
represented as follows: 13
[0060] and a flow chart of a specific example is shown in FIG.
5.
[0061] By way of further illustration of the invention, reference
may be made to the following specific examples:
EXAMPLE 1
[0062] Synthesis of Combretastatin A-4 Prodrugs via Troc
Phosphorylation Route
[0063] Cis-combretastatin A-4 (5 g, 15.8 mmol, leq) was dissolved
in acetonitrile (50 ml) under argon atmosphere and
dimethylaminopyridine (50 mg, 0.41 mmol) and 2,2,2-trichloro-ethyl
phosphorodichloridate (5.77 g, 21.7, 1.4 eq) were added to the
solution forming the phosphate ester of combretastatin A-4.
Triethylamine (2.3 g, 22.7, 1.44 eq) was added to the mixture
portionwise over 20 minutes. After 30 minutes, TLC confirmed the
completion of the reaction. Zinc/copper amalgam (6.26 g) was added
to the solution and the solution was heated to 40.degree. C. After
30 minutes, 2,4-pentanedione (1.62 g, 16.2 mmol, 1.02 eq) was added
in portions while heating at 40.degree. C. After 1.5 hours, heat
was removed and the reaction was cooled to room temperature.
[0064] The solution was filtered and washed with acetonitrile (25
ml.times.2). Water (50 ml) was added to the filtrate and solution
was cooled on ice bath and a precipitate formed upon cooling. Dowex
ion exchange resin (21 g) was added and ice bath was removed. The
mixture turned to a homogeneous orange color suspension. The resin
was filtered out and the filtrate was concentrated under reduced
pressure to remove most of the acetonitrile. The mixture was
dissolved in ethanol and 50% aqueous sodium hydroxide was added to
bring the pH to 12-14. The mixture was stirred at room temperature
for 30 minutes and filtered with an ethanol rinse (50 ml). In order
to purify the product, the crude combretastatin A-4P (2.42 g) was
dissolved in ml H.sub.2O Methanol 50% (24 ml) and the solution was
filtered to remove any undissolved particles. The solution was then
heated to 35-40.degree. C. for 1 hour. Once the solution cooled
down to 30.degree. C. acetone was added (12 ml). Solution was
allowed to cool to room temperature and stirred for 2 hours. A
second volume of acetone was added and the solution was stirred at
room temperature for 12-16 hours and the product was filtered out
the next day. The cake was washed with 20% H.sub.2O/acetone (4.5
ml) twice and then with acetone (4.5 ml). The isolated solid was
dried in high-vacuum oven overnight at 40.degree. C.
EXAMPLE 2
[0065] Synthesis of Combretastatin A-4 Prodrugs via Benzyl
Phosphorylation Route
[0066] Cis-combretastatin A-4 (250 g, 791 mmol, leq) was dissolved
in acetonitrile (1250 ml). Triethylamine (120 g, 1186 mmol, 1.5 eq)
and carbon tetrabromide (320 g, 965 mmol, 1.22 eq) were added to
the solution. Dibenzylphosphite (249 g, 949 mmol, 1.2 eq) was
dissolved in acetonitrile (500 ml). Reaction was cooled to
0.degree. C. and the dibenzylphosphite solution was added dropwise
to the reaction mixture. After one hour, the completion of the
reaction was verified by TLC and HPLC. Distilled
bromotrimethylsilane (TMS-Br) (306 ml, 2373 mmol, 3 eq) was added
to the same mixture. After 30-45 minutes, TLC confirmed completion
of the debenzylation, the reaction was quenched with sodium
methoxide (25 w % in methanol, 560 ml, 2373 mmol, 3 eq) and allowed
to stir overnight. The all cis product was filtered out and washed
with 2.times.400 ml 50% methanol/acetone.
[0067] Crude combretastatin A-4P was isolated in approximately 75%
yield (85% w/w assay). In order to purify the product, the crude
combretastatin A-4P (260 g) was suspended in H.sub.2O (1300 ml ) .
Material dissolved as pH was adjusted to 10-12, using sodium
methoxide/methanol (25 w %). Methanol was added to the solution
(1300 ml) and the solution was filtered to remove any undissolved
particles. The solution was then heated to 35-40.degree. C. for 1
hour. Once the solution cooled down to 30.degree. C. acetone was
added (1300 ml). Solution was allowed to cool to room temperature
and stirred for 2 hours. A second volume of acetone was added and
the solution was stirred at room temperature for 12-16 hours and
the product was filtered out the next day. The cake was washed with
20% H.sub.2O acetone (445 ml) twice and then with acetone (445 ml).
The isolated solid was dried in high-vacuum oven overnight at
40.degree. C. Combretastatin A-4P was isolated in 40% total yield
from starting phenol.
[0068] .sup.1H NMR D.sub.2O, .delta.3.58 (s, 6H), 3.62 (s, 3H),
3.73 (s, 3H), 4.71 (s, 2H) , 6.31 (dd, 2H) , 6.70 (quart, 2H), 7.28
(s, 1H). .sup.13C NMR, D20 .delta.58.52, 58.74, 63.57, 109.13,
114.96, 124.11, 125.22, 131.45, 132.83, 132.92, 136.35, 138.47,
146.03, 151.84, 151.92, 154.77, pH 8.1-8.5. Na 10.1%. HPLC (AUC)
100%, HPLC (w/w) +99 .
EXAMPLE 3
[0069] Comparison of Products
[0070] The Compound V product obtained by the specific process
example represented by FIG. 5 (herein "Process B") was tested and
compared with another sample of combretastatin A-4 disodium
phosphate prepared by an earlier and different process (herein
"Process A") not embraced within the present invention. It will be
noted that Process B (embodying the method of the invention) is
also represented by Example 2 above.
[0071] The absolute identity (actual disposition of atoms within a
unit cell) of these two materials could not be established in the
absence of any single crystal x-ray diffraction data on the
disodium salt. However, the two materials (Compound V obtained by
Process B of the invention, and comparative combretastatin A-4
disodium phosphate obtained by Process B) exhibited physical
differences as characterized by DSC, TGA, powder-XRD and solution
state .sup.13CNMR. Results, set forth in Table 4 below, indicate
that the product of Process B (of the invention), i.e., Compound V,
is a novel product. In Table 4, the comparative product is
designated "Process A Product" while Compound V is designated
"Process B Product."
4TABLE 4 Comparison of Process A and Process B Products Property
Process A Product Process B Product Appearance White powder White
powder Solvent of Ethanol Acetone/methanol/water crystalliza- tion
Microscopy Irregularly shaped plate- Agglomerates of irregularly
like particles shaped needle-like particles DSC Endotherm at
110.degree. C. with Endotherm at 122.degree. C. with a a shoulder
at 77.degree. C. (loss shoulder at 74.degree. C. of volatiles)
Endotherm (loss of volatiles) max at 267.degree. C. Endotherm max
at 258.degree. C. (melting and (melting and decomposition)
decomposition) TGA Weight loss at 150.degree. C. = Weight loss at
150.degree. C. = 4% 6.2% Moisture Loss on drying at 1% RH = Loss on
drying at 1% RH = sorption at 5.7% 6.6% 25.degree. C. Wt. gain at
30-70% RH = Wt. gain at 30-70% RH = 7-8.6% 3% Wt. gain at > 70%
RH = Wt. gain at > 70% RH = 9-30% upto 30% Powder Low
crystallinity Low crystallinity, X-ray Changes X-ray pattern on
different pattern diffaction exposure to different RH from that of
Process conditions A material Lost its X-ray pattern at extremely
low humidity and 100% RH Aqueous pH Solubility(mg/mL) pH
Solubility(mg/mL) solubility* 0.6 2.27 0.80 0.21 at 25.degree. C.
as 0.8 0.83 0.97 0.25 a function of 1.2 0.35 2.20 1.61 pH, pH 5.2
77.4 3.26 5.35 adjusted 5.6 76.8 6.96 34.7 with HCl 7.1 169 7.28
81.2 7.2 204 7.35 94.3 7.8 208 8.40 115 9.4 213 (in water) 9.40 118
(in water) Solubility in Methanol = 22.4 mg/mL Methanol = 1.77
mg/mL organic Ethanol = 0.18 mg/mL Ethanol = 0.096 mg/mL solvents*
at Acetone = 0.45 mg/mL Acetone = BQL.sup.$ 25.degree. C.
*solubility values based on cis-CA4P free acid .sup.$BQL = below
quantitation limit of the HPLC method used
[0072] The powder-XRD patterns and the DSC and TGA thermograms of
these materials were distinctly different from each other. Also,
the initial moisture content for Process B material at the time of
analysis was higher than that for Process A material. The Process B
product showed consistently lower solubility than the Process A
product in both aqueous and organic solvents at 25.degree. C.,
implying greater stability for the Process B product. The Product A
product showed a greater degree of hygroscopicity than the Product
B product. Solution state NMR studies showed that there were no
chemical differences between the two materials. Based on the
available data, it was concluded that Process B afforded a
physically more stable material than Process A. Although there are
physical differences between the two products, the lyophile drug
product prepared from Process B material was in no way compromised
for its quality and stability; in fact is was demonstrated to be
better than the drug product obtained from Process A material.
[0073] It can be appreciated that other salt forms of
combretastatin A-4P may be formed by replacing sodium methoxide
solution with reactive amounts of alkaline metals or inorganic
salts such as Na.sup.2+, Na.sup.+, Li.sup.+, Mg.sup.2+, Mn.sup.2+,
Zn.sup.2+, Ca.sup.2+, Cs.sup.2-, imidazole, morpholine, piperazine,
piperidine, pyrazole, pyridine, adenosine, cinchonine, glucosamine,
quinine, quinidine, tetracycline, or verapamil resulting in salt
forms of combretastatin A-4P with varying solubility.
[0074] An advantage of the subject invention is the phosphorylation
of the combretastatin A-4 in a continuous process, thereby
shortening the reaction from three steps to one step eliminating
time consuming and costly work-ups, isolations, purifications, and
evaporations.
[0075] A further advantage of the subject invention is the
development of improved phosphorylation with the benzyl group
providing an alternative phosphorylation method to the Troc Method
thereby avoiding heavy metal contaminants associated with the
deprotection of the Troc group.
[0076] A further advantage of the subject invention is the
replacement of the ion exchange chromatographic separation of the
phosphate acid with an ion exchange resin.
[0077] A further advantage of the subject invention is the
elimination of carbon tetrachloride, chloroform, DMF and pyridine
from the phosphorylation reaction.
[0078] A further advantage of the subject invention is the increase
in concentration of the reactants thereby allowing increased
loading and increasing yield of combretastatin A-4.
[0079] A further advantage of the subject invention is elimination
of the evaporation of the solvent after the completion of the
reaction.
[0080] A still further advantage of the subject invention is the
elimination of the side products and remaining starting materials
during the wash.
[0081] The subject invention further provides the advantage of a
high throughput, scalable process by eliminating the use of ion
exchange chromatography, hazardous and inconvenient solvents and
expensive reagents, and by increasing the loading in every step.
Consequently, the methods disclosed herein can be scaled up to
produce large quantities of combretastatin A-4 prodrugs.
[0082] The foregoing is a description of a new, useful and
non-obvious method of synthesizing combretastatin A-4 prodrugs.
[0083] It is to be understood that the invention is not limited to
the features and embodiments hereinabove specifically set forth,
but may be carried out in other ways without departure from its
spirit.
* * * * *